CN103083658B - A kind of vaccine adjuvant composition being used for the treatment of or preventing pig infectious disease - Google Patents
A kind of vaccine adjuvant composition being used for the treatment of or preventing pig infectious disease Download PDFInfo
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Abstract
The invention provides a kind of vaccine adjuvant composition being used for the treatment of or preventing pig infectious disease, containing following component: Radix Astragali extract, polymer and antigenic component; Or containing Radix Astragali extract, polymer, cyclodextrin and antigenic component.Described vaccine adjuvant of the present invention can with pig circular ring virus antigen or the antigen combined use of mycoplasma hyopneumoniae, the immune efficacy of vaccine can be increased substantially, as the consumption that at least can be reduced to Proantigen can reach original immune efficacy; On the other hand, proved by the test of the vaccine adjuvant of multiple dosage, vaccine adjuvant of the present invention does not have obvious side effect, and particularly relative to conventional oil phase adjuvant, side effect obviously reduces.
Description
Technical field
The present invention relates to a kind of vaccine adjuvant, refer to a kind of vaccine adjuvant composition being used for the treatment of or preventing pig infectious disease containing astragalus polysaccharides and polymer thereof especially.
Background technology
Vaccine adjuvant is used to reach two objects: it slows down antigen and disengages from injection site, and its stimulating immune system.Traditional vaccine is usually by deactivation or to be killed or the thick prepared product of modified pathogenic microorganism alive forms.The impurity relevant to the culture of these pathogenic microorganism can be used as the adjuvant strengthening immunne response.But add adjuvant tolerable and use the antigen of smaller dose to stimulate similar immunne response, thus reduce the manufacturing cost of vaccine.Therefore, when the effect by some injectable vaccine significantly can be increased when vaccine and adjuvant combination.
Many factors must be considered when selecting vaccine adjuvant.Vaccine adjuvant should make disengaging with absorption rate of antigen slow in an efficient way, and minimum to the toxicity of host, allergenicity, stimulation and other undesired effect.In order to make people satisfied, adjuvant should be non-virucidal properties, the high-caliber immunity of biodegradable, sustainable generation, can stimulate cross-protection, can compatible with plurality of antigens, in multiple species effectively, to host nontoxic and safety (as: without injection site reaction).Other adjuvant characteristics expected be can micro-administration, dosage save, there is excellent shelf stabilities, drying can be born, can be made into not oil-containing, can be used as solid or liquid and exist, to manufacture and its manufacture is not expensive for isotonia, easily.Finally, adjuvant can be configured to according to the needs of vaccination protocols induce body fluid or cellullar immunologic response or the two to have concurrently by high expectations.But, the limited amount of the adjuvant of above-mentioned condition can be met.
Summary of the invention
In view of this, main purpose of the present invention is to provide a kind of vaccine adjuvant composition being used for the treatment of or preventing pig infectious disease, it is characterized in that, containing following component: Radix Astragali extract, polymer and antigenic component.
Preferably, Radix Astragali extract of the present invention contains the astragalus polysaccharides being greater than 70%wt; More preferably, containing the astragalus polysaccharides being greater than 90%wt
Preferably, in Radix Astragali extract of the present invention, astragalus polysaccharides amount is 0.1 ~ 100mg/ml; More preferably, in described Radix Astragali extract, astragalus polysaccharides amount is 1 ~ 10mg/ml; Most preferably, in described Radix Astragali extract, astragalus polysaccharides amount is 3 ~ 4mg/ml.
Preferably, polymer of the present invention is polyacrylic acid; More preferably, described polymer is the polyacrylic acid that commodity are called carbomer (Carbomer); Be more preferably Carbomer940, Carbomer974P, Carbomer934P and Carbomer971P, most preferably be Carbomer934P.
The amount of polymer of the present invention is generally 0.001mg/ml ~ 50mg/ml, preferably 0.1 ~ 10mg/ml, more preferably 0.1 ~ 5mg/ml, most preferably 1mg/ml ~ 3mg/ml.
Described vaccine adjuvant of the present invention and following vaccine antigen are with the use of bacterial antigen or viral antigen; Preferred antigens pig bacterial antigen and viral antigen; Most preferably pig circular ring virus antigen or mycoplasma antigen.
Another aspect of the invention is the preparation method that above-mentioned vaccine adjuvant composition is provided, comprise the steps:
A) in buffer, prepare the compositions of antigenic component;
B) described astragalus polysaccharides Radix Astragali extract is added in the compositions of steps A;
C) described polymer is added in the compositions of step B.
Another object of the present invention is to provide a kind of vaccine adjuvant composition, it is characterized in that, also comprise cyclodextrin derivative.
Preferably, cyclodextrin derivative of the present invention is the cyclodextrin derivative SL-CD being disclosed in CN 1175264A, and its content is 0.1 ~ 10mg/ml, more preferably 0.1 ~ 5mg/ml, most preferably 2mg/ml.
Similarly, the above-mentioned vaccine adjuvant containing cyclodextrin derivative can with following vaccine antigen with the use of bacterial antigen or viral antigen; Preferred antigens pig bacterial antigen and viral antigen, most preferably pig circular ring virus antigen or mycoplasma antigen.
Another aspect of the invention is the preparation method of the vaccine adjuvant composition providing above-mentioned cyclodextrin derivative as SL-CD, comprise the steps:
A) in buffer, prepare the compositions of antigenic component;
B) described Radix Astragali extract is added in the compositions of steps A;
C) described polymer is added in the compositions of step B;
D) described cyclodextrin derivative is added in the compositions of step C.
As seen from the above, be of the present inventionly used for the treatment of or prevent the vaccine adjuvant composition of pig infectious disease to have following advantage at least:
1. can improve vaccine immunity protection, be free from side effects: can be found out by subsequent embodiment of the present invention, vaccine adjuvant of the present invention can increase substantially the immune efficacy of vaccine, as the consumption that at least can be reduced to Proantigen can reach original immune efficacy; On the other hand, proved by the test of the vaccine adjuvant of multiple dosage, vaccine adjuvant of the present invention does not have obvious side effect, and particularly relative to conventional oil phase adjuvant, side effect obviously reduces.
2. vaccine adjuvant preparation of the present invention is simple, directly mixes;
3. the vaccine adjuvant of the application of the invention, greatly can reduce the use amount of antigen in vaccine, thus reduce costs.
Detailed description of the invention
Term:
Radix Astragali extract: the extract of leguminous plant Radix Astagali or the pod membrane Radix Astragali, main component is astragaloside and astragalus polysaccharides.
SL-CD: the cyclodextrin derivative prepared according to China Patent Publication No. CN1175264A embodiment 6; by at least one but no more than N-l to have at least the linear alkyl chain group of 8 carbon atoms and at least one but a no more than N-l sulfate group replace; wherein said linear alkyl chain group is selected from alkyl acyl and alkenylacyl; wherein said linear alkyl chain group and the merging sum of sulfate group are no more than N, are further that wherein N is the hydroxyl value of the cyclodextrin therefrom obtaining cyclodextrin derivative.
" antigen " or " immunogen " refers to the material that any immune stimulatory is replied.This noun comprises killed, deactivation, attenuation or modified antibacterial alive, virus or parasite.Term antigen also comprises the polynucleotide of other or its any combination, polypeptide, recombinant protein, the peptide of synthesis, protein extract, cell (comprising tumor cell), tissue, polysaccharide or lipid or its fragment.Term antigen also comprises antibody, such as anti-experimenter's homologous idiotypic antibody or its fragment, and can the synthetic peptide mimotope of analogue antigen or antigenic determinant (epi-position)
" vaccine " refers to the suspension of one or more killed antibacterials that the component that can be used as vaccine or immunogenic composition uses.
Adjuvant is normally used for reaching two kinds of objects: slow down antigen from the release of injection site and stimulating immune system.
" buffer " refers to the chemical system preventing the chemical system of the concentration change of another kind of chemical substance from referring to the concentration change preventing another kind of chemical substance, as: proton donor and receptor system can be used as the buffer preventing hydrogen ion concentration (pH) from obviously changing.Another buffer example is the solution of the mixture containing weak acid and salt (conjugate base) or weak base and salt (conjugate acid) thereof
" antibody " refers to the immunoglobulin molecules that can be combined with specific antigen because of the immunne response to antigen.Immunoglobulin by the serum proteins having " constant " and " variable " district " gently " and " weight " polypeptide chain and form, and divides into several class (as: IgA, IgD, IgE, IgG and IgM) according to the composition of this constant region
Viral dilution amount required when " TCID50 " refers to " TCID " and be defined as the cell culture through inoculation of infection 50% given batch.Multiple method can be used to calculate TCID50, comprise the Spearman one Kappa method (Spearman-Karber method) used in this specification.
Porcine circovirus 2 type (Porcine circovirus type2 selected in the embodiment of the present invention, PCV2) strain is PCV2b strain SH strain, carry out preservation at China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date: on March 4th, 2008, preserving number is CGMCC No.23890.This strain is open at the open CN101240264 of Chinese patent.
Inactivator of the present invention is formalin, and its final concentration used is 0.1 ~ 0.3% (v/v), and inactivation time is 24 ~ 48h.Preferably, employ the formalin-inactivated PCV2 SH strain of final concentration 0.2% volume in the embodiment of the present invention, inactivation time is 18 hours.
Employ in the embodiment of the present invention Mi Libo (Millipore company) film bag (molecular retention amount is 300Kda) do 3 times concentrate, as subsequent embodiment proves, the method and concentration can make finally to obtain bigeminy vaccine and obtain preferably vaccine effect; The present invention can also use other hyperfiltration process, as cellulose ultrafiltration and the repeatedly method such as ultrafiltration.
For making the present invention easier to understand, below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention, NM specific experiment method in the following example, conveniently experimental technique carries out usually.
Embodiment 1, containing the vaccine adjuvant composition of pig circular ring virus antigen and vaccine thereof
1. the preparation of pig circular ring virus antigen
1.1 produce the preparation of planting with bacterium (poison)
The preparation of porcine circovirus 2 type SH: by seed culture of viruses with MEM culture medium (
invitrogen company) do 10 times of dilutions, PK15 (ATCC is inoculated in by 5% volume, preserving number is CCL-33) cell culture, 37 DEG C adsorb 30 minutes, add the MEM cell maintenance medium of the D-glucosamine hydrochloric acid containing 4% calf serum and 2mmol/L, cultivate 4 for 37 DEG C, freeze thawing 2 ~ 3 times, results virus, virus titer is 10
6.5tCID
50/ ml.
The cultivation preparation of 1.2 virus liquids or bacterium liquid
The preparation of porcine circovirus 2 type SH strain virus liquid: use rolling bottle cell culture method.To the PK15 cell (purchased from ATCC) of monolayer be covered with, remove cell culture fluid, by seed culture of viruses liquid by 0.1 ~ 0.2TCID
50the inoculum concentration an of/cell is inoculated on PK15 cell, gently Spin cells bottle 2 weeks, and 37 DEG C adsorb 30 minutes, add cell maintenance medium, put the cultivation of 37 DEG C of rotations (10 ~ 12 turns/hour).Observe 1 ~ 2 every day, Growth of Cells is good, cultivates harvestings on the 4th and Cell sap, freeze thawing 3 times for 37 DEG C, and put less than-20 DEG C and preserve results virus, virus titer is 10
6.5tCID
50/ ml.
The process of 1.3 virus liquids or bacterium liquid
The process of porcine circovirus 2 type SH strain virus liquid: virus liquid doughnut is filtered post (10 μm, aperture with 0.45 μm) and filters, remove cell debris.
The deactivation of 1.4 virus liquids or bacterium liquid
The deactivation of porcine circovirus 2 type SH strain virus liquid: the virus liquid that the step 1.3 be up to the standards processes is added formalin.Deactivation, makes the final concentration of formalin be 0.2% (V/V), fully shakes up intensification immediately, timing is started when temperature rises to 37 DEG C, keep deactivation in 18 hours complete, the sodium pyrosulfite adding 0.2% (w/v) stops deactivation, puts 2 ~ 8 DEG C of preservations
2. the preparation of Radix Astragali extract
Milkvetch Root is ground into most coarse powder, add 10 times amount water soakings after 1 hour, reflux, extract, 3 times, amount of water is followed successively by 10 times, 8 times, 6 times, extraction time is followed successively by 2 hours, 1.5 hour, 1.5 hour, merge 3 extracting solution, be cooled to room temperature, high speed centrifuge is centrifugal, 8000 rpms centrifugal 20 minutes, supernatant concentration to relative density is 1.15 ~ 1.20, adding dehydrated alcohol to alcohol content is 70%, 4 DEG C leave standstill 12 hours, abandoning supernatant, precipitation is dissolved in water, concentration is made to become the solution of 1g crude drug/ml, adding dehydrated alcohol to alcohol content is 35%, high speed centrifuge is centrifugal, 8000 rpms centrifugal 20 minutes, get supernatant, adding dehydrated alcohol to alcohol content is 70%, 4 DEG C leave standstill 12 hours, abandoning supernatant, precipitation uses 80% successively, 95%, after absolute ethanol washing dehydration, 80 DEG C of vacuum dryings 12 hours, obtain Radix Astragali extract, the content recording astragalus polysaccharides with phenol-sulfuric acid colometry is 90%wt with glucose meter, with high effective liquid chromatography for measuring, it contains astragaloside is 0.01%wt.
3. the preparation of immunologic adjuvant compositions
3.1 preparation storing solutions
3.1.1, the preparation of PBS buffer solution; 0.01M pH=7.2 PBS 2000ml
A: first prepare first liquid: the Na of 0.2M
2hPO
4buffer solution 1000mlNa
2hPO
4.12H
2o 71.625g, water for injection 1000ml, standardize solution in 1000ml volumetric flask
B: prepare second liquid again: the NaH of 0.2M
2pO
4buffer solution 1000mlNaH
2pO
4.2H
2o 31.202g, water for injection 1000ml, standardize solution in 1000ml volumetric flask
C: first liquid: second liquid=72: 28 (volume);
D: first liquid and the mixing of second liquid, and with water for injection by after its 20 times of volume dilution, add by final concentration 0.85% PBS that namely NaCL obtains 0.01M pH=7.2.After subpackage, 121 DEG C of 30min autoclavings.Room temperature is placed.
3.1.2 the preparation of Radix Astragali extract solution
Radix Astragali extract prepared by step 2 to dissolve in deionized water, make stock solution 112 DEG C of 30min autoclavings of 250mg/ml (mass/volume), 4 DEG C of preservations
3.1.3 the preparation of carbomer solution
Carbomer Carbomer 934P (Tian Liyuan bio tech ltd, Qingdao) is dissolved in deionized water, makes stock solution 12 DEG C of 30min autoclavings of 25mg/ml, 4 DEG C of preservations
3.1.4 the preparation of SL-CD solution
Prepare SL-CD according to China Patent Publication No. CN1175264 embodiment 6, with deionized water, make stock solution 12 DEG C of 30min autoclavings that concentration is 0.75%, 4 DEG C of preservations.
3.2 containing the preparation of pig circular ring virus vaccine combination of vaccine adjuvant of the present invention:
Use the Radix Astragali extract solution of above-mentioned preparation, carbomer solution, SL-CD solution, prepare pig circular ring virus vaccine combination according to the methods below:
A) in 10mlPBS liquid, above-mentioned pig circular ring virus antigen liquid is added;
B) Radix Astragali extract solution is added in step A solution;
C) carbomer solution is added in step B solution;
D) SL-CD solution is joined in step C solution;
E) PBS liquid is added to final volume 50ml.
Wherein, according to the form below omission can add the steps such as SL-CD solution, namely omit step D, configure pig circular ring virus vaccine combination as follows:
A) in 10mlPBS liquid, above-mentioned pig circular ring virus antigen liquid is added;
B) Radix Astragali extract solution is added in step A solution;
C) carbomer solution is added in step B solution;
D) PBS liquid is added to final volume 50ml.
Or according to the form below formula omits other compositions, configuration pig circular ring virus vaccine combination:
The pig circular ring virus vaccine containing vaccine adjuvant of the present invention of test example 1-9 is prepared, wherein containing the vaccine adjuvant composition described in above-described embodiment 1 by the formula of table 1 below:
Table 1, pig circular ring virus vaccine combination formula form
The preparation of 3.3 test examples 10 (the pig circular ring virus vaccine of oil-containing adjuvant)
According to oily adjuvant prepared by the method for patent CN101549155A embodiment 3, antigen amount 10.0ml (with test example 1), oil phase 40ml.Method is: joined by oil phase in the clean beaker of 200ml, open mulser switch, stirring at low speed.By the antigen liquid 10ml of test example 1, slowly add in oil phase, slowly accelerate to 450r/min, fully emulsified 10min.
Wherein oil phase is add aluminium stearate in the mixed liquor of white oil 98% (weight/volume) white oil and 2% (weight/volume), and wherein the addition of aluminium stearate is 1g/100ml oil adjuvant mixed solution.
The immune effect contrast of embodiment 2, test example 1-10
1. test animal and grouping
With 5-6 (PCV2 ELISA antibody titer is not higher than 1: 50) 60 in PCV2 ELISA negative antibody Healthy female cleaning grade Balb/c mice in age in week, is divided into 12 groups, often organizes 5.Carry out immunity every 0.2ml according to table 1, after two weeks, carry out the 2nd inoculation by identical approach and dosage; Blank unavoidably.The each group of equal isolated rearing of mice is observed.Two exempt from blood sampling in latter 3 weeks, separation of serum, measure PCV2 ELISA antibody titer in serum.Diluted by its 6400X, 12800X after the blood serum sample mixing of 5 white mice, each dilution factor repeats 2 holes
Table 2 tests grouping and disposition table
2. white mice antibody test
Use PCV2 ELISA antibody testing method: the PCV2 recombinant Cap protein of use escherichia coli expression or the PCV2 of purification are as antigen, being diluted to final concentration with the carbonate buffer solution of pH value 9.6 is 5 μ g/ml, coated elisa plate, every hole 100 μ l, 2 hours are acted at putting 37 DEG C, spend the night at putting 2-8 DEG C: wash 3 times, each 3-5 minute; Every hole adds 200 μ l and closes containing the confining liquid of 0.15%BSA, acts on 2 hours at putting 37 DEG C; Wash 3 times, each 3-5 minute; Serum PBS to be checked is done 1: 50 dilution, and then do 2 times of serial dilutions, each sample a line, every hole 100 μ l, at putting 37 DEG C, act on 1 hour; Wash 3 times, each 3-5 minute; Add the SPA (1: 10000 dilution) of HRP labelling or HRP labelling sheep anti-mouse igg (1: 5000 times of dilution), every hole 100 μ l, effect 1 hour at putting 37 DEG C; Wash 3 times, each 3-5 minute; Add substrate solution TMB to develop the color, every hole 100 μ l, lucifuge colour developing 10-15 minute under room temperature; Add stop buffer (2mol/L H
2sO
4solution), every hole 100 μ l, inherent microplate reader read in 10 minutes the OD value at 450mn wavelength place.Result judges: be judged to the positive with dilution factor serum OD450 value to be checked/negative serum OD450 value (P/N value) when being not less than 2.1.Serum greatest dilution when being not less than 2.1 using P/N value is as the ELISA antibody titer of this serum.Diluted by its 6400X after being mixed by the blood serum sample of same group 5 white mice, each dilution factor repeats 2 holes, is detected by sample in same ELISA Plate.
3. the result of the test of white mice antibody test
The antibody test result of table 3, experimental group
Result of the test: the level of antibody is (experimental group) 6 > 5 > 1 > 4 > 2 > 3 > 9 > 7 > 10 > 8 > 11.
Data acquisition chi-square statistics: result of the test: 1 ~ 10 experimental group is relative to blank group 11 significant difference, 1 ~ 7 and 9,10 experimental grouies are relative to experimental group 8 significant difference not adding Radix Astragali extract or cyclodextrin and polymer, and 1 ~ 6 experimental group is relative to experimental group 9 significant difference.Therefore, use the immunologic adjuvant compositions of Radix Astragali extract of the present invention and carbomer solution and Radix Astragali extract, carbomer solution, SL-CD solution immunologic adjuvant compositions relative to matched group and the immunologic adjuvant compositions effect only containing Radix Astragali extract better, wherein, the effect containing the immunologic adjuvant compositions of Radix Astragali extract, carbomer solution, SL-CD solution is best.
4, vaccine side effect test:
Use injection site Histopathologic change to observe vaccine side effect, often group is got 3 white mice and is observed, concrete steps:
Sampling: drop into neutral formalin more than the internal fixtion 24h of 10% after tissue is in vitro in 15min.
Rinse: piece of tissue be placed in dehydration hand basket, flowing water spends the night flushing from bottom to top.
Dewater transparent: 70% ethanol 60min-80% ethanol 60min-90% ethanol 60min-95% ethanol I 45min-95% ethanol 45min-dehydrated alcohol 30min-dehydrated alcohol 30min dimethylbenzene I 20min-dimethylbenzene II.
Waxdip: the total time 2-h of waxdip.
Embedding: or tangent plane should be specified downwards to imbed in dewaxing by the largest face of piece of tissue during embedding.
Section: the thickness of section is generally 3-5 μm.
Roasting sheet: microscope slide is placed on the baking box interior baking 1-2h of 58-60 DEG C.
HE dyes: dimethylbenzene I 15min-dimethylbenzene II 5min-dehydrated alcohol 5min-dehydrated alcohol 5min-95% ethanol 5min-80% ethanol 5min-70% ethanol 5min-distilled water 5min-hematoxylin 5min-washing-hydrochloride alcohol differentiation 10s-tap water orchidization 30min-Yihong 3min-70% ethanol 5min-80% ethanol 5min-95% ethanol 5min-dehydrated alcohol 5min-dehydrated alcohol 5min-dimethylbenzene I 5min-dimethylbenzene II 5min-neutral gum sealing.
The pathological change of tissues observed under optical microscope: inflammation part observes inflammatory exudate (inflammatory cell, edematous fluid etc.).
To be designated as "-" without inflammatory reaction and obvious pathological change in skin tissue cell;
Be designated as "+" to have a small amount of inflammatory cell and slight pathological change in skin tissue cell;
Be designated as " ++ " to have more inflammatory cell and obvious visible pathological change in skin tissue cell;
Be designated as " +++ " to have a large amount of inflammatory cells and serious pathological change in skin tissue cell.
Table 5, injection site Histopathologic change observed result
Result of the test: result of the test shows, adds oily adjuvant group, has obvious side reaction, and other group does not have side reaction substantially.
Embodiment 3, containing the vaccine adjuvant composition of mycoplasmal pneumonia of swine antigen and vaccine thereof
1. the preparation of mycoplasmal pneumonia of swine antigen
Strain: select strain mycoplasmal pneumonia of swine to be MR48 strain, China Committee for Culture Collection of Microorganisms's common micro-organisms center within 19th, is deposited in JIUYUE in 2006, depositary institution is called for short: CGMCC, deposit number: CGMCCNo.1816, Classification And Nomenclature: mycoplasma hyopneumoniae, English name is Mycoplasma hyopneumoniae, depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, the breeding of Institute of Microorganism, Academia Sinica 1. first order seed: get mycoplasma hyopneumoniae MR48 strain freeze-drying lactobacillus respectively, dilute with fluid medium, be inoculated on solid medium plate, put 37 DEG C and cultivate 7-10d, the bacterium colony that growth selection is good, purely after the assay was approved, as first order seed.
The formula (by 1065ml) of fluid medium: Cor Bovis seu Bubali leachate 300ml, ddH
2o360ml, corrects pH value to 7.4,121 DEG C of sterilizings 15 minutes.Add the composition of following filtration sterilization again: Hank ' s balanced salt solution (10 ×) 40ml, 0.25% (W/V) phenol red 10ml horse serum 200ml, 5% (W/V) lactoalbumin hydrolysate 100ml, 25% (W/V) yeast leachate 20ml, 10000IU/ml penicillin 10ml, 1% (W/V) thaliium acetate solution 25ml
Getting first order seed is respectively inoculated on fluid medium, cultivates 4-7d for 37 DEG C, through purely after the assay was approved, as secondary seed.
The 2 seedlings preparation of bacterium liquid
Respectively mycoplasma hyopneumoniae MR48 strain and seed liquor are inoculated in fluid medium with 1: 10 (volume ratio).Cultivate 4-7 day at depositing 37 DEG C, culture declines more than 0.5 pH value, purely after the assay was approved, then amplification culture to 200 in the same way, 000ml.
3. the deactivation, concentrated and join Seedling of bacterium liquid:
3.1. the assay of deactivation and antigen:
Respectively culture is counted by PCR method.10 times of volume ratio dilution institute inspection cultures, be then PCR, the minimum limitation that PCR detects is 3 × 10
-3μ g (being equivalent to about 1000 thalline), calculates thalline number (Shen Qingchun, Tan Qingsong by the multiple of dilution, Wang Qin, Mhp culture bacterium number [J] is measured Deng .PCR method. Chinese Preventive Veterinary Medicine report, 2006,28 (1): 55-57).Culture content is 1 × 10
9mHDCE/ml.(MHDCE=mycoplasma hyopneumoniae DNA cell equivalents, namely 1MHDCE is equivalent to 1 mycoplasma hyopneumoniae).
3.2 concentrate:
Mycoplasma hyopneumoniae culture fluid deactivation be up to the standards and mycoplasma hyorhinis culture fluid are used Mi Libo (Millipore company) film bag (molecular retention amount is 300Kda dalton) to do 10 times respectively and are concentrated.
3.3 join Seedling:
Use the Radix Astragali extract of embodiment 1, carbomer solution and SL-CD, the compound method containing the porcine mycoplasmal vaccine combination of vaccine adjuvant of the present invention is as follows:
A) in 10mlPBS liquid, antigen is added;
B) Radix Astragali extract is added in step A solution;
C) carbomer solution is added in step B solution;
D) SL-CD solution is joined in step C
E) PBS liquid is added to final volume 50ml
Press the porcine mycoplasmal vaccine combination of formulated containing vaccine adjuvant of the present invention of table 6 below, i.e. test example 11-14.
Table 6, porcine mycoplasmal vaccine combination prescription form
4. test example 11-14 effect test:
Select 21 ~ 25 age in days ablactational baby pig 40, be divided into 4 groups, often organize 10 (seeing the following form); 1st, 2,3,4 groups of every pigs musculi collis injection i (mycoplasma hyopneumoniae) vaccines respectively, every 2ml, the 5th group contrast as counteracting toxic substances, do not inoculate; , neither inoculate, also not counteracting toxic substances, raise in independent room.1st, 2,3,4 groups after inoculation four months with strong malicious F65 strain (purchased from China Veterinary Drugs Supervisory Inst.) counteracting toxic substances of mycoplasma hyopneumoniae.After counteracting toxic substances (DPC) 30 days, no pain puts to death all pigs.Take out lung, become by the demographic's pneumonopathy not understanding experimental group.Inoculation the same day (0DPV), inoculate latter one month (1MPV), inoculate latter four months (4MPV) or the counteracting toxic substances same day (0DPC), counteracting toxic substances after 30 days (30DPC), collect blood sample from all pigs, use competitive ELISA test kit (being manufactured by DAKOCo) to detect the serum Hangzhoupro body of anti-mycoplasma hyopneumoniae.Described blood serum sample is kept at-20 DEG C until test.
Table 7, the process of porcine mycoplasmal vaccine combination prescription
5. lung lesion score and result
Under appraisal result is shown in:
The pneumonopathy of table 8, porcine mycoplasmal vaccine combination prescription becomes score result
| Group | The quantity of pig | Average lung lesion score (%) |
| 1 | 10 | 3.6 |
| 2 | 10 | 4.2 |
| 3 | 10 | 3.7 |
| 4 | 10 | 7.3 |
| 5 | 10 | 15.2 |
It is 15.2% that the average pneumonopathy of matched group 5 becomes, and it is 3.6% that the average pneumonopathy of vaccine group 1 (test example 11) inoculation group becomes, and it is 4.2% that the average pneumonopathy of vaccine 2 groups of inoculation group (test example 12) becomes.It is 3.7% that the average pneumonopathy of vaccine 3 groups (test example 13) inoculation group becomes, and it is significant difference between 7.3%. inoculation group and matched group that the average pneumonopathy of vaccine 4 groups (test example 14) inoculation group becomes.This shows: the protective immunity of vaccine energy effective stimulus opposing mycoplasma hyopneumoniae described in test example 11,12,13, and immune effect is suitable.
Result of the test shows: at the experimental group of antigen amount by few half, add immunologic adjuvant compositions of the present invention, still can reach original immune effect.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (12)
1. be used for the treatment of or prevent a vaccine combination for pig infectious disease, it is characterized in that, described compositions is composed of the following components: Radix Astragali extract, polymer and antigenic component, and wherein, described polymer is polyacrylic acid.
2. vaccine combination according to claim 1, is characterized in that, described Radix Astragali extract contains the astragalus polysaccharides being greater than 70%wt.
3. vaccine combination according to claim 1, is characterized in that, described Radix Astragali extract contains the astragalus polysaccharides being greater than 90%wt.
4. vaccine combination according to claim 1, is characterized in that, in described Radix Astragali extract, astragalus polysaccharides amount is 0.1 ~ 100mg/ml.
5. vaccine combination according to claim 1, is characterized in that, described polyacrylic acid is Carbomer 934P.
6. vaccine combination according to claim 1, is characterized in that, the amount of described polymer is 0.001mg/ml ~ 50mg/ml.
7. the vaccine combination being used for the treatment of or preventing pig infectious disease, it is characterized in that, described compositions is composed of the following components: Radix Astragali extract, polymer, cyclodextrin derivative and antigenic component, wherein, described polymer is polyacrylic acid, described cyclodextrin derivative is SL-CD, and content is 0.1 ~ 10mg/ml.
8. a preparation method for the vaccine combination as described in claim 1-4 and 6 any one, is characterized in that, comprise the steps:
A) in buffer, prepare the compositions of antigenic component;
B) described Radix Astragali extract is added in the compositions of steps A;
C) add in the compositions of step B by described polymer, wherein, described polymer is polyacrylic acid.
9. a preparation method for vaccine combination as claimed in claim 5, is characterized in that, comprises the steps:
A) in buffer, prepare the compositions of antigenic component;
B) described Radix Astragali extract is added in the compositions of steps A;
C) add in the compositions of step B by described polymer, wherein, described polymer is polyacrylic acid Carbomer 934P.
10. a preparation method for vaccine combination as claimed in claim 7, is characterized in that, comprises the steps:
A) in buffer, prepare the compositions of antigenic component;
B) described Radix Astragali extract is added in the compositions of steps A;
C) add in the compositions of step B by described polymer, wherein, described polymer is polyacrylic acid;
D) described cyclodextrin derivative is added in the compositions of step C.
11. according to the vaccine combination of claim 1-7 any one, and it is characterized in that, described antigenic component is bacterial antigen or viral antigen.
12. according to the vaccine combination of claim 1-7 any one, and it is characterized in that, described antigenic component is pig circular ring virus antigen or mycoplasma hyopneumoniae antigen.
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